Chimeric invasin system

Abstract

A transkingdom platform for the delivery of therapeutics to target cells. The system maintains the export and uptake functions of Inv while modifying its targeting away from β1 integrin to other proteins expressed on the surface of target eukaryotic cells (i.e., a cell surface protein) or chemical moieties (i.e., a cell surface chemical moiety) expressed on the surface of a target eukaryotic cell by replacing D4 and D5 of Inv with a binding domain from a heterologous protein via genetic engineering. These heterologous proteins could be derived from bacterial, fungal, animal, or viral genomes. This engineering would result in the construction of a chimeric Inv protein in which D1-D3 (i.e., the non-binding domains) are fused in frame to an alternative binding domain derived from a heterologous protein. The alternative binding domain would interact with a different cell surface protein or chemical moiety, which can in some instances be referred to as a receptor, on the surface on the surface of a eukaryotic cell, thereby allowing specific targeting to cells independent of Inv's intrinsic β1 integrin binding.

1 . An expression cassette for the production of a chimeric invasin (Inv) polypeptide comprising a prokaryotic promoter and a nucleic acid sequence encoding an Inv polypeptide fused to a linker polypeptide at the carboxy terminus of the Inv polypeptide and wherein expression of the nucleic acid encoding the chimeric invasin polypeptide is controlled by the prokaryotic promoter. 
     
     
         2 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  further comprising a sequence encoding a binding domain wherein the binding domain is fused to the amine terminus of the linker polypeptide. 
     
     
         3 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 2  wherein the binding domain is a binding domain from a protein listed in Tables 1-3 or the binding domain is a synthetic binding domain. 
     
     
         4 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the nucleic acid sequence of the invasin region of the chimeric polypeptide is 90% identical to nucleic acids 1-2958 of SEQ ID. NO. 37. 
     
     
         5 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the nucleic acid sequence of the invasin region of the chimeric polypeptide is 95% identical to nucleic acids 1-2958 of SEQ ID. NO. 37. 
     
     
         6 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the nucleic acid sequence of the invasin region of the chimeric polypeptide is 99% identical to nucleic acids 1-2958 of SEQ ID. NO. 37. 
     
     
         7 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the nucleic acid sequence of the invasin region of the chimeric polypeptide is 90% identical to nucleic acids 1-2382 of SEQ ID. NO. 37 or SEQ ID. NO. 38. 
     
     
         8 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the nucleic acid sequence of the invasin region of the chimeric polypeptide is 95% identical to nucleic acids 1-2382 of SEQ ID. NO. 37. 
     
     
         9 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the nucleic acid sequence of the invasin region of the chimeric polypeptide is 99% identical to nucleic acids 1-2382 of SEQ ID. NO. 37. 
     
     
         10 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the nucleic acid sequence encodes a linker polypeptide selected from the group consisting of [SEQ. ID. NO. 2] through [SEQ ID. NO. 20] and/or a protease cleavage site selected from the group consisting of [SEQ. ID. NO. 22] through [SEQ ID. NO. 36]. 
     
     
         11 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the nucleic acid sequence encodes a protease cleavage site cleaved by a peptidase or protease. 
     
     
         12 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the nucleic acid sequence encodes a protease cleavage site cleaved by a peptidase or protease listed in Table 4. 
     
     
         13 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 2  wherein the nucleic acid sequence encoding the protease cleavage site cleaved by a peptidase or protease is between the sequence encoding the invasin and the sequence encoding the binding domain. 
     
     
         14 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the prokaryotic promoter is a promoter selected from the group consisting of T7, lacUV5, gapA, T5, recA, Ptac, Patac, pAl, lac, Sp6, araBad, and trp. 
     
     
         15 . The expression cassette for the production of a chimeric invasin polypeptide according to  claim 1  wherein the prokaryotic promoter is a hybrid or synthetic prokaryotic promoter. 
     
     
         16 . (canceled) 
     
     
         17 . The bacterium expressing a chimeric invasin polypeptide according to claim  16   claim 1  wherein the bacterium is a bacterium selected from the group consisting of  Clostridium difficile, Escherichia coli, Clostridium tetani, Helicobacter pylori, Fusobacterium nucleatum, Gardnerella vaginitis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, Vibrio vulnificus, Salmonella typhi, Clostridium botulinum, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium lepromatosis, Corynebacterium diptheriae, Klebsiella pneumoniae, Acinetobacter baumannii, Streptococcus mutans , group  B streptococci, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumonia, Enterococcus spp., Enterococcus faecalis, Listeria, Yersinia, Rickettsia, Shigella, Salmonella spp., Legionella, Chlamydia, Brucella, Neisseria, Burkolderia, Bordetella, Borrelia, Coxiella, Mycobacterium, Helicobacter, Staphylococcus, Streptococcus, Porphyromonas, Vibrio, Treponema, Lactobacillus , and  Bifidobacteriae.    
     
     
         18 . The bacterium expressing a chimeric invasin polypeptide according to  claim 1  wherein the bacterium is an  Escherichia coli  bacterium. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . A chimeric invasin polypeptide comprising an Inv polypeptide and a linker polypeptide, wherein the linker polypeptide has a first end (N-terminus) and a second end (C-terminus), wherein the first end (N-terminus) of the linker polypeptide is attached to the C-terminus of the Inv polypeptide. 
     
     
         22 - 31 . (canceled) 
     
     
         32 . A chimeric invasin polypeptide comprising the D1-D3 domains of the Inv polypeptide attached to the binding domain of a heterologous protein or a synthetic binding domain. 
     
     
         33 - 65 . (canceled)